1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding proteins that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding proteins that confer drought, cold, and/or salt tolerance to plants.
2. Background Art
Environmental stress causes significant crop losses. The stresses are numerous and often crop- and location-specific. They include drought, high salinity, temperature extremes, hypoxia, mineral nutrient deficiency and UV-B radiation. Research in this area is driven by the hope of improving crop yield in afflicted areas. Currently, actual but slow advances are being made by crop breeders and agronomists using tried- and tested methodology. However, biotechnology will increasingly have a role as genes involved in stress tolerance are cloned and their mode of action elucidated (Quarrie S A, 1996 Plant Growth Reg. 20:167–178). By improving a plant's performance in response to different environmental stresses, the losses in productivity and risks to farming can be greatly reduced. Modifying a plant's tolerance to environmental stresses also allows a plant to be grown in regions where a plant or plant variety is typically unable to grow (Bohnert and Jensen, 1996 TIBTECH 14:89–97).
The biochemical and physiological basis for plant stress tolerance is not well understood. However, it does appear that common damage from different stresses (drought, salt and cold stress) is mostly due to dehydration (Smirnoff, 1998 Curr. Opin. Plant Biol. 9:214–219). The response that distinguishes drought-tolerant and -sensitive species most clearly is a dramatic accumulation of osmoprotective substances and also of ions that leads to osmotic adjustments (Bohnert and Jensen 1996 TIBTECH 14:89–97).
Sucrose is an important osmoprotectant under dehydration stress conditions (water deficit and freezing stress). The biosynthesis of sucrose has several control steps (Stitt and Sonnewald, 1995 Ann. Rev. Plant Physiol. Plant Mol. Biol. 46:641–368) and pyrophosphate (PPi) metabolism plays an important role in this biosynthetic pathway (Stitt, 1998 Bot. Acta 111:167–175). PPi is mainly produced as a by-product of biosynthesis of macromolecules such as RNA, proteins and cellulose in elongating cells (Nakanishi and Maeshima, 1998 Plant Physiol. 116:589–597). There are three enzymatic activities involved in metabolizing PPi in the cytosol, two reversible reactions catalyzed by PFP (Pyrophosphate dependent Fructose-6-P Phosphofructokinase; Paul et al., 1995 Planta 196:277–283) and UGPase (UDP-Glucose Pyrophosphorylase; Zrenner et al., 1993 Planta 247–252), respectively, and the tonoplast membrane bound inorganic pyrophosphatase (PPase, Rea et al., 1992 Tr. in Biochem. 17:348–353). A soluble inorganic pyrophosphatase exists only in chloroplasts, but not in the cytosol (Weiner et al., 1987 Biochem. Biophys. Acta 893:13–21).
Two families of soluble inorganic pyrophosphatase are known to date. Family I, including most of the currently known PPases, and family II, including PPases from certain bacterial species. The two families do not show any sequence similarity to each other. In addition to soluble PPases, plants (and certain bacteria) have a membrane-bound PPase that works as a reversible pump. Membrane-bound PPases are much larger and do not have any sequence similarity to any of the two families of soluble PPase. Plant soluble PPase resemble prokaryotic PPase more closely than animal/fungal PPases (Sivual et al., 1999 FEBS Letters 454:75–80).
The ectopic expression of a soluble inorganic pyrophosphatase in the cytosol has been shown to shift carbon metabolism towards the synthesis of sucrose in the transgenic plants due to the removal of PPi as a product of the PFP and UGPase reaction. The constitutive over-expression (35S promoter) of E. coli soluble pyrophosphatase in the cytosol of plant cells leads to an accumulation of sucrose in the leaves of the transgenic tobacco plants (Jellito et al., 1992 Planta 188:238–244). The same effects were obtained upon expression of the enzyme only in the phloem (rolC promoter) indicating the essential role of PPi for phloem loading of sucrose (Lerchl et al., 1995 Plant Cell 7:259–270). Additionally, both the 35S- and the rolC-plants were reduced in growth. These examples show the importance of PPi for plant metabolism and growth. The alteration of the amount and/or availability of inorganic phosphate in the cell also has profound effects on plant lipid metabolism (Haertel et al., 2000 PNAS 97:10649–10654), and thereby can lead to changes in the composition of the plasmalemma as well as the organelle membranes.
The response of transgenic plants expressing a soluble inorganic pyrophosphatase in the cytosol to environmental stresses like drought, salt and freezing has not been determined thus far. Therefore what is needed is the identification of genes and proteins encoded by those genes that have pyrophosphatase-like activity and that are involved in stress tolerance in plants. Elucidating the function of genes expressed in stress tolerant plants will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement. Newly generated stress tolerant plants will have many advantages, such as increasing the range that crop plants can be cultivated by, for example, decreasing the water requirements of a plant species. This invention fulfills in part the need to identify new, unique pyrophosphatases capable of conferring stress tolerance to plants upon over-expression.